Project 1 will focus on aspects of two fundamental processes involved in limb pattern formation, AER-directed outgrowth and patterning of limb mesoderm, and the segmentation of continuous cartilaginous skeletal rudiments into two or more separate elements by joint formation. Many of the cells of the AER continuously undergo apoptosis as the AER is directing limb outgrowth and patterning, suggesting that programmed cell death may play an important role in maintenance of AER activity and signalling. This hypothesis will be tested in part by using an AER- specific enhancer element in the Msx-2 gene to target ectopic expression of Bcl-2, a potent inhibitor of apoptosis, specifically to the AER of the developing limbs of transgenic mice to determine if AER directed outgrowth or patterning of the limb is altered. Msx-2 and BMPs is highly expressed in the ER and have been implicated in regulating apoptosis in light mesoderm, suggesting that Msx-2 and BMPS may be in a regulatory network that controls the extensive programmed cell death that continuously occurs in the AER. To study their functions in the AER and examine their possible roles in apoptosis, and AER-specific enhancer element in the Msx-2 gene will be used to direct the expression of inhibitors of BMP or Msx-2 function to the AERs of transgenic mice. The homeobox-containing gene Cux, the chicken ortholog of the Drosophila Cut gene, is highly expressed at all of the discrete sites of incipient joint formation in the developing limb, as is Gdf5, a BMP family member which has been shown to lay an important role in joint formation. The hypothesis that Cux plays a crucial role in regulating the onset of the segmentation process that generates joints between the skeletal elements of the limb will be investigated, as will the possible relationship between Cux and GDF5 in the process. The onset of joint formation is characterized by the joint conversion of differentiating chondrocytes that express high amounts that express high amounts of cartilage- characteristic type II collagen into the flattened densely packed cells of the joint interzone which express little or no type II collagen. Vertebrate Cut homologs in general are transcriptional repressors that inhibit the expression of tissue specific genes in multiple lineages. Thus the hypotheses that Cox might regulate the onset of joint formation at lest in part by repressing the expression of the type II collagen gene or other cartilage specific genes, thus facilitating the formation of the joint interzone tissue, will be studied.